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At the beginning of the game the player creates a party of six characters from a choice of five classes (Rocketjock, Warrior, Medic, Rogue, and Engineer) and six races (Human, Desert Runner, Tinker, Venusian, Martian, and Mercurian).

The game centers around a longstanding war between Earth and Mars, more specifically between two organizations, the Earth-based New Earth Organization (NEO), and the Mars-based Russo-American Mercantile (RAM). The militaristic and dictatorial RAM, backed by a powerful army of genetically engineered soldiers, has been laying siege to Earth for years. NEO, effectively an organized resistance movement, is forced to operate out of secret bases on the surface of Earth and in orbit in its uphill battle for human freedom.

The game begins as the player-created party joins the ranks of a desperate NEO. After an introductory briefing, the NEO facility is discovered and attacked by RAM rocketships and troop transports. The actual gameplay begins and the player's first goal is to repel the RAM attack. During this first level of play, NEO NPCs join the party during combat with the RAM invaders. When the player successfully locates and activates the anti-aircraft defenses of the NEO base, the RAM attack is finally driven back.

Now considered valuable NEO operatives, the party is summoned to NEO's headquarters, a hidden space station in Earth orbit called Salvation III. Disguised as space junk, Salvation manages to elude RAM detection and serves as the NEO headquarters. For their first mission as a discrete unit, the party is assigned a small shuttlepod and instructed to sweep the nearby area. During the patrol, a derelict spacecraft is discovered and the party is ordered to board and investigate. Upon boarding, the airlock malfunctions and the party's pod disengages the derelict and drifts away, leaving them stranded on the seemingly abandoned spacecraft. Through a search of the derelict, the party realizes that the previous crew had been killed by an infectious brain parasite. The ship is also infested with violent, genetically engineered creatures which randomly (but consistently) attack the party. As the time spent on the derelict grows, members of the party begin to show symptoms of parasitic infection which, if left untreated, causes the infected to go berserk and attack other party members. Additionally, the scavenging creatures begin to evolve into larger (and more powerful) forms. The sole survivor of the ship's original crew, an artificial intelligence (or DP, digital personality) agent named Scot.DOS living in the ship's computers, assists the party in subduing the monsters by flooding the ship with argon gas, which is toxic to the gennies. The infected team members can also be cured with Scot's help—provided they have not already become psychotic. After the brief reprieve, Scot.DOS informs the team that one of the gennies has managed to survive the gas attack and is attempting to initiate a self-destruct sequence. The situation climaxes as the party races up an elevator shaft and combats the last surviving gennie—which has evolved into a humanoid form and is equipped with a relatively heavy armament—and disables the destruct mechanism. With the infection and gennie threats aboard the derelict removed, the party is able to return to Salvation with the salvaged Maelstrom Rider for debriefing and to enjoy the spoils of a mission accomplished.

At this point in the game, the player is sent on a mission to Mars itself, to infiltrate the RAM base, Gradivus Mons. For the first time, the solar system view is used, allowing the player to travel to any of the inner planets or asteroids. While exploring space, the player's ship occasionally encounters other vessels, which by and large are hostile. The player may encounter vessels from the RAM navy, but also occasionally meets space pirates and Mercurians. Enemy vessels may be destroyed in space, or boarded for ground combat.

On Mars, the player meets the Desert Runners, a slender, athletic, warrior-like people who have been genetically engineered with canine and feline attributes in order to more easily adapt to the Martian desert environment, and live without the assistance of most technology. Prior to the meeting, the player becomes aware of an impending RAM attack on the natives' city, but is unable to earn their trust before the attack begins. In a parallel to the beginning of the game, the party must repel the RAM attack, and fights alongside Desert Runner NPCs in combat. The RAM troops engaged in this battle are much tougher (and more numerous) than the NEO attack at the beginning of the game, and growing enemy numbers begin to become a theme of the game. The party earns the trust and respect of the Desert Runners, and their leader, Tuskon, helps the party infiltrate Gradivus Mons and destroy the Doomsday Laser prototype along with the entire base.

After their mission on Mars, the party's ship is unavoidably crippled, boarded, and captured by space pirates. Conveniently, the party meets another prisoner, the titular Buck Rogers and manage to overthrow their captors. Buck then returns with the party to Salvation and assists them (largely through advice and direction) as they travel to a RAM laboratory on Venus to investigate unsettling news that RAM is working on a massive weapons project (i.e. research and support for the Doomsday Laser orbiting Mercury).

Ultimately, the party discovers RAM, along with the corrupt Mercurian government, is building its ultimate weapon, the eponymous Doomsday Laser, in orbit of the planet Mercury with aspirations of destroying Earth entirely. After managing to land on Mariposa Three, an orbital city near Mercury, the party makes its way through a military complex and fights through a series of increasingly difficult battles. Finally finding themselves at the command center for the Doomsday Laser itself, the members of the party successfully destroy the weapon and manage to escape the massive explosion. The defeat severely damages RAM's ability to make war with Earth. NEO is, for the moment, victorious.

Most versions of the game display the Adventuring view from the point of view of the party (i.e. the display is first-person), and have a simple text menu for spaceports.

The Sega Genesis version of the game shows the Adventuring view as an isometric view of the area indicating the characters behind the party leader (i.e. the display is third-person), and uses the Adventuring view in spaceports. Most of the text is replaced by icons.

The available races and classes are also more limited on the Sega Mega Drive/Genesis version. The player may only select from Human, Desert Runner, and Tinker as races, and may select only Warrior, Rocketjock, Medic, or Rogue as a character class. There are also significantly fewer skills and equipment types available to players as compared to the PC version.

Computer Gaming World called the story "very satisfying" and the game "fun to play". It concluded that Buck Rogers was "good, light adventuring and a nice change of pace from the fantasy line".[3] In 1991, Dragon gave the game 4 out of 5 stars.[4]MegaTech magazine praised the absorbing gameplay. Mega placed the game at #39 in their Top Mega Drive Games of All Time.[5]